Method of coating a bioactive substance

ABSTRACT

The invention generally provides a method of coating a material, the method including the following steps: (a) forming a generally elongate coating structure ( 15 ), the coating structure having an internal cavity ( 17 ) extending at least substantially along its length, and wherein the internal cavity of the coating structure is capable of receiving a core material ( 21 ); (b) inserting a core material ( 21 ) into the internal cavity ( 17 ) of the coating structure ( 15 ); (c) compressing the coating structure at a first location ( 44 ) along its length so as generally to form a seal at that location; and (d) compressing the coating structure at a second location along its length ( 45 ).

INTRODUCTION

This invention relates to apparatus and methods for coating a material.As will become apparent from the following description, the inventioncan be used in many coating applications, however one application towhich the invention is particularly suited is in relation to theproduction of coatings for pharmaceutical capsules, tablets and likedevices for the delivery of pharmacologically active substances (foreither human or veterinary use) to a patient. The invention isespecially suited for use in the production of pharmaceutical capsules,tablets and like delivery devices where the coating material for thepharmaceutical delivery device is intended to control or delay thetiming of release of a pharmacologically active material containedwithin the “core” of the device. While noting the invention'ssuitability in a broad range of applications, it will be described inthe following description with particular reference to thepharmaceutical coating application to which it is particularly suited.

BACKGROUND TO THE INVENTION

In order for an effective dosage regimen to be delivered, manypharmaceutical substances must be administered according to exacting oroccasionally, complex dosage regimes. Compliance with such regimes isparticularly important in, for example, many third world countries,where patient compliance with a dosage regimen may be impaired due tothe patient's inability to understand the nature of the dosage regimenrequired, or the need for compliance. Similarly, the treatment ofinfants by a repeated course of injections can be particularly traumaticto the infant patients concerned, and the delivery of the regimen by areduced number of doses whose release profile is controlled isparticularly desirable. Another important scenario where controlledrelease delivery can be particularly advantageous (compared to repeatedapplication of individual doses) is in veterinary medicine. Many animalsreact extremely adversely to the infliction of pain by (for example)repeated injections. In the veterinary treatment of animals, it can beparticularly difficult to ensure that the animal has received the dosagerequired of the pharmaceutical agent concerned (particularly, forexample, if the pharmaceutical agent involved must be administered inminute quantities, such as, for instance, with reproductive hormonesused in animal husbandry). Repeated handling of an animal to administerdrugs to it not only runs the risk that the animal will refuse toco-operate with the handler, and that the required dose of the drug maynot in fact be delivered, but also, it runs the increased risk of injuryto the animal handler that must administer the drugs.

The use of controlled release pharmaceutical delivery devices istherefore particularly desirable in many instances, including thosedescribed above. However, the delivery of drugs via controlled releaseregimens is not as widespread as might be hoped, because the productionof suitable delivery devices for the controlled release ofpharmacologically active substances is presently limited by themanufacturing methods 3 and production apparatus that have been used todate to make them.

Several methods have been employed to date in order to manufacturecontrolled release delivery devices to contain a dose of apharmaceutical agent in the form of (for example) a tablet. One methodthat has been employed is injection moulding. This technique involvesthe injection of a heated coating material (typically, apharmaceutically acceptable polymer) under pressure into a mould for adelivery device (in the form of, for example, part of a container). Oncethe coating material has cooled and solidified to a suitable extent, themould is opened for ejection. The part container must then be filledwith the pharmaceutical agent it is intended to contain, and it mustthen be sealed. This technique has significant limitations, particularlyfor the mass production of controlled release pharmaceutical deliverydevices. For one thing, as explained above, it results in the productionof only a partial container. This means that the process of producing acontrolled release drug delivery device via the injection mouldingmethod is a multi-step procedure, which requires sequentially (i) first,the production of a partial container, (ii) filling the partialcontainer with the required dose of the drug it is intended to contain,and (iii) then sealing the container. This sequential, multi-stepprocedure is inefficient as a manufacturing process. It also involves atime delay between the initial step of manufacturing thepartial-container and the subsequent steps of filling it with thepharmaceutical moiety and sealing the container, which can give rise todifficulties as regards ensuring that the finished product issufficiently sterile for use in human or veterinary medicine.

A second technique is available for manufacturing pharmaceuticaldelivery devices in such a way as to overcome the sterility problemsdescribed earlier that affect the injection moulding procedure. In thepharmaceutical context, to date, the second technique appears to havebeen used solely in the manufacture of sterile vessels to containliquids like physiological saline or water for injection. This secondtechnique is referred to in the art as the “blow-fill-seal” (or the“form-fill-seal”) method. In this technique, a polymer (usually aplastics material) is melt-processed and extruded from a die to form alength of tube. When extruded, the tube is sufficiently hot to bemalleable, but not so hot as to be liquid, and therefore, so as to beuncontrollable in the subsequent steps involved in the manufacturingprocess. A multi-piece die (containing a mould which encompasses thelength of extrudate) then clamps around the length of extrudate (whichis known as the “parison”), thereby sealing one end of the parison, andleaving the other end gripped by the die, but slightly open. The parison(which at this stage of the process, remains hot and malleable) is athin tube which is suspended within the mould cavity. Air is theninjected into the interior of the parison so as to inflate it, so thatit assumes the shape of the mould. The inflated parison is then filledwith the desired contents by an injection process, and is then sealed.The die is then opened to release the finished product. The“blow-fill-seal” technique is similar in many respects to injectionmoulding. It is therefore subject to at least some of the same problemsthat apply to the injection moulding technique.

The present invention aims to avoid one or more difficulties associatedwith the prior art manufacturing techniques described above, and theapparatus used to perform them.

GENERAL DISCLOSURE OF THE INVENTION

The invention generally provides a method of coating a material, themethod including the following steps:

(a) forming a generally elongate coating structure, the coatingstructure having an internal cavity extending at least substantiallyalong its length, and wherein the internal cavity of the coatingstructure is capable of receiving a core material;

(b) inserting a core material into the internal cavity of the coatingstructure;

(c) compressing the coating structure at a first location along itslength so as generally to form a seal at that location; and

(d) compressing the coating structure at a second location along itslength.

The method may be performed sequentially in the order of steps (a) to(d) set out above, or the steps of the method may be performed inanother sequence. A preferred sequence is (from first to last step):

Step (a) (first step)

Step (c) (second step)

Step (b) (third step)

Step (d) (fourth step).

Preferably, the step of forming the coating structure involves extrudingit from an extrusion means. In this embodiment of the invention, thecoating structure must be an extrudable material, such as a plasticsmaterial which is amenable to processing techniques such asmelt-processing and extrusion when heated. The extrusion means could forexample, take the form of a die which extrudes the coating structure inlengths of any desired cross-sectional shape. Preferably, thecross-sectional shape of the coating structure formed by the extrusionmeans is circular, so that in its three-dimensional configuration, thecoating structure generally has the appearance of a tubular structure.The coating structure's cross-sectional shape could take any desiredform however, including generally square, triangular, elliptical, ovoidor more complex shapes. The proviso here is that the cross-sectionalshape must be a closed shape (such as the shapes described earlier), sothat the extruded coating structure can be sealed when it is subjectedto the compression steps of the method, described earlier. Thecross-sectional shape adopted for the coating structure in any contextmay depend on the application to which the coated material produced bythe coating method is to be put.

In one form of the invention, the coating structure comprises a singlelayer of the material from which it is formed. In other forms of theinvention however, the coating structure may contain two or more layersof the same or of different materials from which it is formed. Formingthe coating structure from two or more layers may be preferred where itis desired to control in a particular manner, the release profile of thecore material from a finished container produced by the method. Byforming the layers of the coating structure from materials havingdifferent degradation or permeability characteristics, a differentialrate, onset or profile of release of the core material into asurrounding environment, as desired, could therefore be established.

Preferably further, in step (d) of the method, a seal is formed at thelocation of the second compression along the length of the coatingstructure, so as to define a closed container which comprises thecoating structure sealed at two opposed ends, and containing the corematerial in its internal cavity. Preferably, the seal at each end of thecontainer is formed by compressing two mutually opposed surfaces of thecoating material so that they come into contact with one another in asealing manner. A seal at any location could also be formed in otherways however, such as by heating the coating structure in addition tocompressing it at the location concerned. The step of compressing thecoating structure at any location could also involve cutting the coatingstructure at each of the first and second locations along its length, soas to define a free-standing container separated from the remainingcoating structure from which the container was formed. Preferablyfurther, such a container would have the appearance of a capsule, suchas that of a conventional pharmaceutical capsule.

In some applications, it might be desirable to produce a container whichis sealed at one end, but not at the other. In such a case, in step (d)of the method, the step of compressing the second location along thelength of the coating structure could take the form of simply cutting itby using a cutting means, rather than forming a seal at that end. Thisversion of the method might be used so as, for example, to form apharmaceutical delivery device having one sealed end and one “open” end,so as to provide a delivery device which in use, would encourage theegress of the core material into a patient, from the “open” end of thedevice.

In other applications, it may be preferred to form a container which iscompletely unsealed at one end, and is “generally” sealed at the other.By “generally” sealing the container at the other end (meaning the firstlocation along the length of the coating structure, in the descriptionset out herein), and further, by reference in step (c) of the firstaspect of the invention, mentioned earlier, to “generally forming aseal”, it is to be understood that a closure which falls somewhat shortof a full or complete seal, is also comprehended. Thus, the formation ofa partial seal at that location would be embraced by this feature of theinvention.

Preferably further, the method would be suitable for continuousoperation, or for repeated operation, along the length of the coatingstructure. In this way, the method C could be used to generate a desirednumber of individual containers each containing a length of the coatingstructure which are sealed as desired, and each of which contains anamount of the core material in its internal cavity. It is even furtherpreferred that automation means be provided to operate the method, sothat a convenient integrated method is provided for producing a desirednumber of containers as described above.

In a particularly preferred embodiment of the invention, the method isused to produce pharmaceutical delivery devices, such as coatedcapsules, coated tablets and the like. In this embodiment of theinvention, the coating structure takes the form of a pharmaceuticallyacceptable substance or combination of such substances. Typically, thecoating will comprise a pharmaceutically acceptable polymer orco-polymer, such as a plastics material. Particularly preferredsubstances for the coating material include polylactide-co-glycolidepolymers, polyesters, polysaccharides, polyamides, poly (amino acids),poly (ortho esters), polyanhydrides, polyphosphoesters and polymersformed through combinations of chemical bonds (such as pseudo-peptides,poly (phosphoester-urethanes) and polydepsipeptides). The core material,in this embodiment of the invention, contains the pharmacologicallyactive substance (for convenience called the “bioactive” in theremainder of this specification) whose release profile is desired to becontrolled by the provision of the coating structure. The bioactive canbe any suitable pharmacologically active substance. Typically, thebioactive would be a hormone or a vaccine, although the method isapplicable to producing pharmaceutical delivery devices suitable for abroad range of bioactives, including natural, synthetic or recombinantpharmacological agents, food additives or food supplements, antigens,antibodies, cytokines, growth promotants, hormones, cancer cellinhibitory agents, immuno-suppressants or immuno-stimulants,anti-microbial agents (including antibiotics), anti-viral agents,vitamins, vaccines, minerals, and organic or inorganic nutrients. Abioactive core material for use in the invention may consist of one typeof the aforementioned substances, or may include combinations of two ormore such substances.

The bioactive (or a composite core material in which it is contained)could also take any number of physical forms, such as in the form of atablet, a gel, a paste, as granules, in powder form, or a fluid, as wellas others. The core material could also contain additional materials tothe bioactive itself, including pharmaceutically acceptable carriers andexcipients (including dispersion media, coatings, antibacterial,anti-fungal and/or anti-viral agents and the like, as well as salts suchas di-calcium phosphate), inert (and pharmaceutically acceptable)materials designed to control further the release of the bioactive in adesired manner, and other like materials, as desired. For example, thecore material could also contain a hydrophilic material to encourage theentry of water into the device (such as for example, a swelling agent,such as a “hydrogel”) or a substance affecting the osmotic interactionof the core material with an external biological fluid. Additionalcomponents could include:

(i) binders, such as gum tragacanth, acacia, corn starch or gelatine;

(ii) disintegrating agents, such as corn starch, potato starch, alginicacid and the like;

(iii) lubricants, such as magnesium stearate;

(iv) explosive combinations (eg, citric acid/sodium carbonate);

(v) surfactant materials or other surface active molecules (eg,proteins, such as albumins, biological detergents and tweens);

(vi) solubility enhancers (eg, sodium citrate, sodium bicarbonate,magnesium carbonate);

(vii) absorbance enhancing agents;

(viii) lubricants;

(ix) flow promoters;

(x) plasticisers;

(xi) antisticking agents: and/or

(xii) anti-static agents.

As would be apparent to those skilled in the art, in pharmaceuticalapplications of the invention, all such additional components must be atleast substantially pharmaceutically pure, non-toxic in the amountsused, and biocompatible with the bioactive(s) used and with the coatingmaterial.

The invention also provides an apparatus for coating a material, theapparatus including:

(a) means for forming a generally elongate coating structure having aninternal cavity extending at least substantially along its length, andwherein the internal cavity of the coating structure is capable ofreceiving a core material;

(b) means for inserting a core material into the internal cavity of thecoating structure;

(c) means for compressing the coating structure at a first location Qalong its length so as generally to form a seal at that location; and

(d) means for compressing the coating structure at a second locationalong its length.

Preferably, the means for forming the elongate structure are extrusionmeans, as discussed earlier.

As explained above, the core material could take any number of physicalforms, such as in the form of a tablet, a gel, a paste, as granules, inpowder form, or as a fluid, as well as others. The means for insertingthe core material into the coating structure could therefore take anynumber of forms, according to the nature of the core material to beinserted into the coating structure. For example, where the corematerial takes the form of a gel or paste, the means for inserting itinto the coating structure could take the form of a nozzle designed toinject the core material into the internal cavity of the coatingstructure.

The means for compressing the coating structure at the first locationcould, for example, take the form of a mechanism for simply pressing twoopposed surfaces of the coating structure together. Such means couldalso include means for forming a cut through the coating structure atthat location. The means for compressing the coating structure at thesecond location could also take a similar form, however they could, ifdesired, take the form of a mechanism for forming a non-sealing open cutat the second location, so as to form a capsule with an open end at thesecond location.

Preferably, the apparatus is suitable for continuous, or repeatedoperation along the length of the coating structure, so as to lenditself to mass production of segments of a coated material, in the formof capsules or like devices.

The invention further provides a coated material produced by a processwhich includes the steps of:

(a) forming a generally elongate coating structure, the coatingstructure having an internal cavity extending at least substantiallyalong its length, and wherein the internal cavity of the coatingstructure is capable of receiving a core material;

(b) inserting a core material into the internal cavity of the coatingstructure;

(c) compressing the coating structure at a first location along itslength so as generally to form a seal at that location; and

(d) compressing the coating structure at a second location along itslength.

Preferably, the coated material produced by the process takes the formof a coated tablet, coated capsule or a like device. Preferably furtherthe coated structure is suitable for use as a controlled release devicefor delivering an amount of the core material in a predetermined manner.It is particularly preferred that in such a device, the core materialcontains a pharmacologically active substance, such as any of thebioactives described earlier. The process could also be configured toproduce a coated material containing multiple units of the samebioactive, or a two or more units of different bioactives.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described by way of example only withreference to the following drawings, in which:

FIG. 1a represents a side cross-sectional view of an apparatusconstructed in accordance with the invention, for use in coating amaterial;

FIG. 1b represents a cross section of the apparatus depicted in FIG. 1a,taken along the line A—A, and when viewed in the direction of the arrowB;

FIG. 2 represents a side cross-sectional view of a second apparatus forcoating a material, constructed in accordance with the invention;

FIG. 3 depicts a modified version of the embodiment of the inventionshown in FIG. 2;

FIG. 4 represents a rear loading forming apparatus constructed inaccordance with a fourth embodiment of the invention (being an enlargedview of part of the apparatus depicted in FIGS. 7-9);

FIGS. 5(a) to (c) depict three different embodiments of a formingapparatus for use in the invention;

FIGS. 6(a) to (c) depict three further embodiments of a formingapparatus for use in the invention;

FIGS. 7-9 depict sequential stages of a capsule-coating process andsystem utilising a fourth embodiment of the invention;

FIG. 10 is an enlarged schematic view of a coated capsule made inaccordance with the invention; and

FIG. 11 is a graph which charts the results of an experiment performedin rats to measure the controlled release (delay) of a bioactiveformulation made in accordance with the invention, relative to a controlgroup.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Referring now to the drawings, FIG. 1a shows an apparatus (generallydenoted 1) for coating a material, constructed in accordance with afirst embodiment of the invention. Apparatus 1, which is shown in crosssection from the side, is an apparatus which could be used for coatingcapsules, such as pharmaceutical capsules. As can be seen from FIG. 1a,apparatus 1 consists of an extruder barrel (generally denoted 3), havingan internal bore 5, and a threaded extruder screw 7 located andsupported for rotation inside the bore 5, by any suitable means, such asthose that would readily understood by persons skilled in the art.

The extruder barrel 3 includes a cross head die 9 near its distalextremity. In use of apparatus 1, a flowable coating material is placedin bore 5, and by rotating extruder screw 7 about its longitudinal axisD within bore 5 in a direction so as to convey the flowable coatingmaterial towards the cross head die 9, is extruded from an opening 11 onthe cross head die. The cross head die 9 includes an extrusion sleeve13, whose longitudinal axis F is at right angles to the longitudinalaxis D of the extruder screw 7. As shown more particularly in FIG. 1b,extrusion sleeve 13 is of generally circular shape when viewed incross-section, in the embodiment of the apparatus shown. When theextruder screw 7 is rotated within bore 5, this motion causes agitationof the flowable coating material so that it is moved towards and ontothe outer surface 12 of extrusion sleeve 13, from within bore 5.Ultimately, such agitation causes the flowable material to exit from theopening 11 (which, by virtue of the cross sectional shape of extrusionsleeve 13, means that in the embodiment shown in FIGS. 1a and 1 b,opening 11 is a generally annular opening). This in turn results in anelongate, generally tubular coating structure 15, formed from theflowable material, being extruded from the apparatus, as shown in FIG.1a. This generally tubular coating structure can be used to coat amaterial within its internal cavity 17, which generally takes the formof a lumen.

As shown more particularly in FIG. 1a, extrusion sleeve 13 has aninternal bore 19. This internal bore is designed to receive a material21 (which in FIG. 1 is shown as being in the form of a generallycylindrical tablet or pellet) within it, so that material 21 may beconveyed to the end 23 of the extrusion sleeve, where it is enters thecavity 17 and is encapsulated by the generally tubular coating structure15. In the embodiment of the apparatus 1 shown in FIGS. 1a and 1 b, theextrusion sleeve is configured so that the material 21 to undergocoating is fed into the internal bore 19 from an upwardly disposed end25 of the extrusion sleeve, so that it falls within the extrusionsleeve's internal bore under the influence of gravity, so as to beconveyed to and to exit from the lower end of the extrusion sleeve, forcoating by the generally tubular coating structure. In other embodimentsof the invention, the extrusion sleeve might not be so disposed withinthe apparatus, and means other than gravity could be used to convey thematerial 21 to the coating point. Those skilled in the art would readilyunderstand that such means could take any number of forms. In theembodiment of the invention shown in FIGS. 1a and 1 b, the material 21to undergo coating is fed into the generally tubular coating structure15 in the same direction as the egress of that structure from theapparatus 1. In this specification, an arrangement whereby the directionof feed of the coating material is the same as the direction of egressof the coating structure from the general forming apparatus is termed“rear loading”.

A different embodiment of an apparatus for coating a material and acoating method in accordance with the invention is shown in FIG. 2. Inthe description that follows, for ease of reference and comparison,features depicted in the embodiments described hereafter that correspondto similar features depicted in FIGS. 1a and 1 b will be accorded thesame reference numerals. In FIG. 2, the apparatus (also generallydenoted 1) also includes an extruder barrel 3, having an internal bore 5and an extruder screw 7, as in the first embodiment of the inventiondiscussed earlier. However, in this embodiment, the end of the extruderbarrel takes the form of a tube die 27, rather than a cross head die. Inthis embodiment, the flowable coating material is extruded from anopening 11 located at the end of the apparatus 1, so that it is extrudedin the direction of the arrow G shown in

FIG. 2, that is, in a direction generally parallel to the longitudinalaxis H of the apparatus 1. The opening 11 from which the flowablecoating material is extruded is also circular in shape, when viewed inthe direction of the arrow J shown in FIG. 2. Opening 11 is an annularhole defined by a circular aperture in the end wall 29 of the apparatus1, and by a circular forming means 31 located within that circularaperture. The circular aperture 29 and the forming means co-operate sothat the flowable coating material is extruded as a generally tubularcoating structure from the opening 11. A material 21 to be coated ispositioned ahead of the direction of flow of the coating structure 15 soas to be fed into, or is fed into the internal cavity 17 of thatstructure by movement in a direction opposite to the direction of egressof the coating structure 15 from apparatus 1. In this specification,such an arrangement for coating a material with the coating structureformed within apparatus 1 is termed “front loading”.

A further, slightly modified version of a front loading coatingapparatus constructed in accordance with the invention, is shown in FIG.3. The apparatus is substantially the same as that depicted in FIG. 3,except that it also includes the use of an additional forming means 33,which in the embodiment shown, takes the form of a generally tubularstructure which is adapted to enter the cavity 17 of the generallytubular coating structure 15. A material 21 to be coated is introducedinto the lumen 35 of the tubular forming means 33, and conveyed by anysuitable means (such as, for example, by an injection of air into thelumen at the opposite end of the forming means 33) to place the material21 within the coating structure, so as to coat the material. The formingmeans 33 would desirably be readily retractable from the tubular coatingstructure 15.

The embodiments of the method described above each involve the initialstep of forming a generally elongate coating structure (in the form ofthe generally tubular coating structure 15), via a forming means.Generally, an extrusion process would be best suited for this purpose,although other methods of forming the coating structure may be used inthe invention. Where an extrusion process is used, the material used toform the coating structure must be amenable to manipulation by processessuch as melt-processing, so that the material can be suitably extrudedfrom the forming apparatus to give rise to the coating structure in thedesired conformation. For pharmaceutical applications, suitablematerials would generally include pharmaceutically acceptable polymers,such as pharmaceutically acceptable plastics. Examples ofpharmaceutically acceptable polymers would includepolylactide-co-glycolide polymers, polyesters, polysaccharides,polyamides and poly (amino acids), poly (ortho esters), polyanhydrides,polyphosphoesters and polymers formed through combinations of chemicalbonds (such as pseudo-peptides, poly (phosphoester-urethanes) andpolydepsipeptides). Other suitable materials could be used, the natureof which would be apparent to those skilled in the art.

In each of the embodiments described earlier, the method involves thesecond step of inserting the core material into the internal cavity ofthe coating structure. The nature of the core material will vary inaccordance with the nature of the application to which the invention isto be put. In pharmaceutical applications, the core material willgenerally contain a bioactive. A broad range of hormones, including bothsteroid and non-steroidal hormones, and many types of vaccines, wouldconstitute suitable bioactives for this purpose.

In pharmaceutical applications of the invention, the bioactive wouldoften be included as part of a composite core material, which could takeany number of physical forms. For example, the bioactive could becontained in the form of a tablet, a gel, a paste, as granules, inpowder form, as well as combinations of the foregoing, and otherphysical forms. In pharmaceutical applications, the core material wouldoften contain additional substances to the bioactive itself, includingpharmaceutical excipients, inert (and pharmaceutically acceptable)materials which might be designed to control the release of thebioactive in a particular manner, and other like materials, as desired.Examples of additional materials which could be contained within thecore material include hydrophilic materials to encourage the entry ofwater into a coated pharmaceutical delivery device (such as for example,swelling agents, such as a “hydrogel”) could be used for this purpose,as could substances affecting the osmotic interaction of the corematerial with an external fluid (such as a biological fluid). Additionalcomponents include:

(i) binders, such as gum tragacanth, acacia, corn starch or gelatine;

(ii) disintegrating agents, such as corn starch, potato starch, alginicacid and the like;

(iii) lubricants, such as magnesium stearate;

(iv) explosive combinations (eg, citric acid/sodium carbonate);

(v) surfactant materials or other surface active molecules (eg,proteins, such as albumins, biological detergents and tweens);

(vi) solubility enhancers (eg, sodium citrate, sodium bicarbonate,magnesium carbonate);

(vii) absorbance enhancing agents;

(viii) lubricants;

(ix) flow promoters;

(x) plasticisers;

(xi) antisticking agents; and/or

(xii) anti-static agents.

As explained earlier, and as would be apparent to those skilled in theart, in pharmaceutical applications of the invention, all suchadditional components must be at least substantially pharmaceuticallypure, non-toxic in the amounts used, and biocompatible with thebioactive(s) used and with the coating material.

The means employed for inserting the core material into the internalcavity of the coating structure will also depend upon the nature of thecore material adopted in any particular application. Generally, themeans for inserting the core material into the coating structure willinclude a chute or nozzle into which the core material is introduced,and by which it is inserted into the coating structure. Such means couldalso include apparatus for pushing the core material into a desiredposition within the coating structure (eg, an injection of air to conveythe core material to, or to position the core material in place in thecoating structure). If the core material is a tablet, it could bedeposited into position with, for example, a pushrod which retractsclear of the coating structure prior to its compression by a compressionmeans. Where the core material in the form of a liquid, paste, granulesor in a powder form, the means for inserting it into the coatingstructure may take the form of an injection apparatus to inject it intothe coating structure as appropriate. The injection mechanism used wouldbe designed so as to be retractable from the coating structure prior toany compression step occurring.

Where the coating structure has been formed by melt-processing andextrusion, it may be necessary to cool the extrudate so that itsolidifies to a suitable extent to enable the core material to be loadedinto it, and at the same time, not damaged. Any suitable means (such asthose that would be apparent to persons of ordinary skill in the art)could be used for this purpose. It is particularly desirable that thecooling processes cool the extrudate at such a rate so that when thetime comes for the core material to be inserted into it. It has cooledto a sufficient extent to be able to receive the core material safely.

The third step of the production method involves the step of compressingthe coating structure at a first location along its length so asgenerally to form a seal at that location. The purpose of compressingthe coating structure is to compress, seal and (where appropriate), tocut the coating structure at the point where compression occurs.Examples of apparatus that could be used to perform the firstcompression step are depicted in FIGS. 5(a) to (c) and FIGS. 6(a) to(c). The apparatus depicted in FIGS. 5(a) to (c) take the form of a“scissors” apparatus (generally denoted 35, which includes a pair ofblades 37 adapted to slide across one another. The widest section of thecompressed area along the coating structure should not exceed theoutside diameter of that structure.) Thus, the provision of notches(such as the notches 39 shown in FIGS. 5(a) to (c)) would ensure thatthe area of compression on the coating structure is not greater insection than the cross-sectional diameter of that structure.

The means for compressing the coating structure at either the firstand/or second locations in the process could alternatively take the formof a “plier” mechanism, where two blades meet one another. FIGS. 6(a) to(c) depict three examples of possible mechanisms for achieving such acompression action. As can be seen, in FIGS. 6(a) to (c). one blade(denoted 40 in each of those drawings) includes a concave recess 41,whereas its opposed blade includes a mating protrusion 43. In use ofsuch plier apparatus in conjunction with the invention, after the stepof inserting the core material into the coating structure has occurredin the production method, the filled coating structure would be passedthrough the concave part of the plier means, and the two blades would bebrought together so as to cut through the filled coating structure. Byvirtue of the mating fit of the two blades, the cut formed would nothave a sectional diameter exceeding the cross-sectional diameter of thecoating structure.

A third possible compression means would take the form of a plier actionwith two blades, where one or both blades have a cutting edge which isnot sharp. Such a mechanism would simply compress, rather than cut thefilled coating structure, and in some applications may be necessary, incombination with a cutting means, to achieve a seal on the filledcoating structure. Whichever form of compression apparatus is used, itcould either be free-standing (and capable, if desired, of beingmanually operated), or more desirably for automated mass productionsystems, it would be integrated into, and form part of an integratedapparatus for carrying out the method aspects of the invention.

When the filled coating structure is compressed so as to cut it at twolocations along its length, a separated segment of the filled coatingstructure is formed. As shown in FIG. 10, in a preferred embodiment ofthe invention, the segment formed by this method includes a sealed outerlayer (consisting of the coating structure (15), which is sealed uponitself at each of points 44 and 45, so as to define a capsule whichincludes a core material 21, within its internal cavity 17. The sealingof the capsule formed by this process could also be achieved by meansadditional to those described above (eg. by heating the capsule at eachof its ends).

Desirably, the manufacture of sealed containers (such as the oneschematically depicted in FIG. 10) would be produced by an automated orpartly automated process capable of being performed on a continuous orrepeated basis, so that many such sealed containers could beconveniently produced by operating the process. Such a process isdepicted schematically in FIGS. 7-9. As shown in FIG. 7, a coatingstructure 15 (which when viewed in three dimensions, would have agenerally tubular structure) is extruded from a forming apparatus 1(shown in enlarged view in FIG. 4) onto a conveyor belt 47. Multipleunits of a core material 21 (which might take the form of apharmaceutical tablet) are rear loaded into the internal cavity 17 ofthe extruded core structure 15, via a sleeve 25. As shown in FIG. 8, afirst compression means 49 compresses the coating structure 15 betweeneach of the locations where a unit of the core material 21 has beendeposited. This causes opposed surfaces of the coating structure 15 tobe brought together, by effectively “pinching” them. This first step ofcompression may occur shortly after the coating structure 15 exits theforming apparatus 1, so as to ensure that the core material 21 isproperly retained within the coating structure. This might be importantwhere, for example, the coating structure 15 is extruded downwardly fromthe forming apparatus, so that if compression of the coating structurewere not to occur at that point, the coating material would fall out ofthe coating structure, under the influence of gravity. Such an Qarrangement is depicted in FIG. 1.

As shown more particularly in FIG. 9, a subsequent compression is thenperformed on the extruded coating structure, at a second location, by asecond compression means 51. In the process depicted in FIG. 9, thesecond compression means takes the form of an apparatus which bothcompresses the coating structure, and at the same time cuts it, so as toform individual capsules (each denoted 53), which are conveyed by theconveyor belt to be further processed as desired according to theparticular application to which they are to be put. Such furtherprocessing might, for example, take the form of heating the ends of thecapsules so as to seal them more effectively, if so required for anyparticular application. (4)

Many variations of the process described above can of course be made,without departing from the general concept of the invention. Forexample, an automated processing system embodying the inventive conceptcould include more than two compression/cutting mechanisms, to achieveproduction efficiencies. Further, the profile of the filled coatingstructure could be collapsed between individual units of the corematerial contained within it, by restricting the ingress of air. Thiscould be achieved, for example, by limiting the ingress of air into thecavity of the coating structure at predetermined times in theperformance of the process (typically, between the insertion of units ofthe core material). This might aid the process of sealing individualcoated structures. The cutting and sealing of loaded sections from alength of extrudate could be achieved with a blade (either cold orheated), a saw, a pinching mechanism, or a non-mechanical cutter, suchas a laser. The appropriate cutting and sealing mechanism(s) would bechosen to suit the particular application.

Co-ordination of the various process steps involved in producing coatedstructures such as those described above could be achieved by anysuitable means, including mechanical means (eg where the movement of allmoving parts is mechanically synchronised), or by electro-mechanicalmeans (where the motion of moving parts is controlled electronicallyfrom sensors appropriately positioned to detect the status of thevarious stages of the process). Hydraulic or pneumatic components usedin the apparatus and process could conveniently be controlled in thismanner. In many modem manufacturing plants, the process wouldadditionally be controlled by computerised means.

The sealed structures produced by the method and apparatus aspects ofthe invention could take many forms, and could be produced in a broadvariety of sizes and dimensions (including conceptually, even micro andeven nano-particulate sizes). Such containers could be put to a broadvariety of uses, and it is to be understood that invention is by nomeans limited to the pharmaceutical applications which have beendiscussed earlier in this specification. For example, sealed containersmade in accordance with the apparatus and method aspects of theinvention could be used to encapsulate transponders for injection intoanimals, for veterinary identification. Further, such coated structurescould also be used for producing controlled release devices forreleasing agricultural chemicals into the environment (eg, forfertilising the growth of plants). It is therefore to be understood thatthe invention is capable of many modifications and/or variations, andthat the spirit and scope of the invention is by no means limited to thedetails of the preferred embodiments described earlier. It is thereforeto be understood that the spirit and scope of the invention extends toevery novel feature and combination of features disclosed herein.

EXAMPLES

The following non-limiting examples of the invention are given below.

A. In Vitro Experiments

In a first set of experiments, the applicants prepared controlledrelease containers in accordance with the method aspect of theinvention, and measured the latency of release from those containers, ofa bioactive release “marker” (Methylene Blue) in an in vitro settingapproximating typical physiological conditions in a mammalian species.The details of the experiments conducted and the results obtained fromthem are set out below.

In the experiments, four (4) batches of “Resomer” coated tablets,approximately 25 mm in length and of 3 mm external diameter wereproduced for testing, as follows. (It should be noted that the term“Resomer” is a trade mark of the Boehringer Ingelheim corporate group).

Materials Used:

Polymers

Polymer formulations used were:

1 R206 (as supplied by manufacturer);

2 RG858 (as supplied by manufacturer);

3 RG506 (as supplied by manufacturer); and

4 RG504 (as supplied by manufacturer).

(The “manufacturer” was the Boehringer Ingelheim corporate group).

Resomer poly-d,l-lactide, R206 (i.v.=1.0; 200 g: Batch #241888) wasobtained from Boehringer Ingelheim.

Resomer 85:15 poly-d,l-lactide-co-glycolide copolymer, RG858 (i.v.=1.4;200 g: Batch #261073) was obtained from Boehringer Ingelheim.

Resomer 50:50 poly-d,l-lactide-co-glycolide copolymer, RG506 (i.v.=0.8;200 g: Batch #34034) was obtained from Boehringer Ingelheim.

Resomer 50:50 poly-d,l-lactide-co-glycolide copolymer, RG504 (i.v=0.4;200 g: Batch #34015) was obtained from Boehringer Ingelheim.

Chloroform (HPLC grade) was from BDH (Hypersolv).

All other chemicals and reagents were generally analytical reagentgrade.

Gel Permeation Chromatography (GPC)

Gel Permeation High Performance liquid chromatographic analysis was usedto confirm the repeatability of production of controlled release devicesusing the apparatus and method aspects of the invention.

The GPC-HPLC system consisted of a Hewlett Packard 1050 series pump andauto-sampler. This was connected with a series of Hewlett-Packard andPLgel columns (polystyrene-divinylbenzene), consisting of one PLgel 10μm mixed-B (#10 μMIXB8434) and two HP/PLgel 5 μm mixed-D columns (#'s5μ48879 and 5μ523413), 300×7.5 mm each, and a PLgel guard column (5 μm;5×100 mm). A Sedex 55 Evaporative Light Scattering detector operating ata temperature of 35 degrees Celsius with nitrogen gas purge/nebuliserwas used for peak detection. The system was run isocratically withchloroform as the mobile phase at a flow rate of 1.0 ml/min.

Samples of polymer extrudate were prepared for molecular weight analysisusing Gel Permeation Chromatography (GPC) by dissolving the sample(about 20 mg) in chloroform (1.0 ml). Samples (400 μl) were then placedin auto-sampler vials containing a Flow Rate Marker (“FRM”; 25 μl),sealed and then run through the HPLC system. The sample injection volumewas 10 μl.

Molecular weights (M_(n) and M_(w)) were calculated from data acquiredby Hewlett Packard Chem Station Software and treated using proprietaryGPC analysis software after setting integration limits manually.Calibration used a series of polystyrene standards (M_(r): 2880000,1290000, 560000, 66000 and 10100 atomic mass units (“amu”)). Thepolydispersities of the standards were 1.03-1.06 for all standards.Standard samples were run subsequent to every sixth or seventh sample.

GPC Data Analysis:

GPC data analysis entailed the following procedure:

raw data files in HPChem Station format were downloaded for importationinto the proprietary GPC analysis software;

the standards from each run were analysed to ensure no anomalousoccurrences had arisen during the chromatographic run. The latter wasdetermined as follows.

comparison of the Peak Volume and Peak molecular weight for each of thestandard samples; followed by

use of the first standard for column calibration to optimise theanalysis algorithm; with

the molecular weights of each of the standards, including the firststandard, then being determined using the calibrated column data.

The molecular weight of the analysis samples was then calculated usingthe column calibration algorithm established from the appropriate groupsof standards.

Extrusion

Processing was performed on a Brabender Extruder at the CASEY Centre forPolymer Technology, Dandenong, Victoria, Australia. The equipment usedconsisted of the following:

Brabender Extruder—Plasticorder Type PL2000-6 consisting of

Dynamometer (Model No. 814400)

Interface (Model No. 680118104)

Temperature Controller (Model No. 680147)

Single Screw Attachment Type 19/25D

Haul Off (Model No. 297828)

Crosshead Die Assembly.

The conditions typically used for extrusion of thed,1-polylactide-co-glycolide polymers are given in Table I below. Theprocedure for setup and extrusion involved the following:

1 When the temperatures had stabilised in the apparatus, the extruderbarrel was purged with Ethylene vinyl acetate (EVA). When the purgematerial appeared clear of any contaminant the barrel was left to runempty.

2 The screw and haul-off belt speeds were then set appropriately for thematerial.

3 Polymer was loaded into the feed throat and pushed, stuffed or starvefed as required.

4 Air cooling of the extrudate was set up as required.

5 The first material through was assumed to be contaminated with purgepolymer and was discarded (approximately the first 50 grams).

6 The resultant polymer extrudate was then hauled off.

The extruder was then run to empty.

TABLE 1 Brabender Extruder Conditions Zone Zone Polymer ID 1° C. Zone 2°C. 3° C. Die ° C. rpm d,l-pLa R206 90 110 125 130 35 d,l-pLa/pGa RG858110  125 140 165 10 (85:15) d,l-pLa/pGa RG506 90 110 125 130 35 (50:50)d,l-pLa/pGa RG504 90 110 125 130 20 (50:50)

The extrusion conditions for these process runs were dependent on theindividual polymer characteristics. However, the data contained in Table1 indicate the conditions generally used.

Each of the four runs progressed relatively smoothly, having stabilizedvirtually immediately following commencement of the extrusion process.It was observed that samples from the beginning to the end of theextrusion run exhibited similar weight average (M_(w)) and numberaverage (M_(n)) molecular weights. The mean molecular weights, relativeto polystyrene, for the selected samples are presented in Table 2.

TABLE 2 Formulation Details Average Molecular Weight of Unit SamplesSelected Samples Code Formulation Prepared M^(n) M^(w) Polydispersity 1Resomer R206 160 47300 120833 2.6 2 Resomer 160 156500  260167 1.7 RG8583 Resomer 160 31850  91808 2.9 RG506 4 Resomer 160  5080  17725 3.5RG504

In Vitro Performance Monitoring

Samples were placed in approximately 20 ml of phosphate buffered saline,(pH 7.2), and held at 37 degrees Celsius. The samples were checkeddaily. Checking involved some agitation of the solution/suspension,albeit not vigorous enough to induce mechanical damage. Release of thebioactive marker from any test sample was determined by visualinspection (namely, by the solution turning blue).

Results:

Polymer Extrusion

Polymer extrusion with the Brabender extruder proceeded underconditions, and in a manner, consistent with materials of comparablecomposition. Each of the four runs progressed smoothly. GPC-HPLC dataindicated that the extrudate exhibited similar weight average molecularweights throughout the runs. The mean molecular weight of the extrudatewas, in general, equivalent to 90% of the molecular weight of thestarting materials indicating minimal thermal degradation had beeninduced.

(B) In Vivo Studies

Outline of Experiments Conducted

The studies set out in A (above) confirm the suitability of controlledrelease devices made in accordance with the invention, in an in vitroenvironment. The applicants also conducted studies to confirm that theinvention could be used to produce devices suitable for use ascontrolled release delivery devices in vivo. These further studies arediscussed below.

The general nature of the experiment was as follows. Rats were randomlyselected into two groups. The first group (Group B) receivedadministration of an Avidin formulation. The second group (Group A) wasadministered the same Avidin formulation, however, presented in the formof a controlled release delivery device made in accordance with theinvention. The time delay to the appearance of antibodies in each groupto the Avidin formulation, was measured. The experimental details wereas follows.

Avidin Formulation

The Avidin formulation used had the following composition:

Avidin 5 mg/35 mg Lactose 95.56% (w/w) Magnesium Stearate 3% (w/w)Explotab (Sodium start glycolate) (1.94% w/w)

Preparation of Implants

Controlled release delivery devices containing the Avidin formulationwere made as follows. Avidin tablets weighing 25 mg were coated using aplastics extrusion apparatus into which 50:50poly-d,l-lactide-co-glycolide copolymer had been melt processed, so asto form individual coated tablets, of approximately 12 mm length and 3.5mm diameter, in accordance with the method aspect of the invention.

Animals

Rats (Sprague-Dawley; Male; 20), mean mass=254 (Stdevp=36 g) wereemployed for the experiment (two randomly selected groups of ten fortesting of response to antigen).

All animals were given free access to food (Barastoc rodent pellets) andwater at all times during the study. All animals were inspected daily bythe animal house supervisor, and thrice weekly for detailed observationby the applicants' personnel to ensure no adverse/traumatic reactions tothe implanting or implants arose.

Animals were identified using permanent colour marker pens and markingthe animals on the tail.

Test Groups

The dose regimens applied to the two experimental groups were asfollows:

Group A: delayed release “Avidin” (10 animals) Group B: “Avidin” only(10 animals)

Implantation

Rats were implanted by means of a modified Synovex® implanting gun.Animals were not anaesthetised. The implantation site was shaven, thenwashed with ethanol (70% v/v) prior to implantation. The implantpuncture wound required no surgical closure.

Subsequent to implantation, all animals were observed for trauma and/orabnormal behaviour for 180 minutes. No abnormal behaviour was observedduring this period. Minimal wound bleeding was observed during theobservation period. Examination of the animals 24 hours afterimplantation showed all wounds had sealed with no signs of infection.

ELISA Testing Protocol

Nunc “Immuno” Microtitre plates were coated with 30 μl of antigen(Avidin stock solution; 10 mg/ml) at 5-50 μg/ml in 0.04 M carbonatecoating buffer. The plates were incubated at 37° C. for 1 hour.

Control test groups of the following composition were run with the testserum:

No antigen, Antibody, Horse Radish Peroxidase (HRP), TMB

No antigen, No antibody, No HRP, TMB

Antigen, No antibody, HRP/TMB

Antigen, known positive antibody, HRP/TMB

Coated plates were washed with water (3×) or saline/Tween 20.

Unbound sites were blocked by incubating for 60 minutes at 37° C. using300 μl/well of 1% BSA in PBS plus 0.1% Tween 20 in PBS.

“Blocked” plates were washed (3×) with saline/Tween 20.

Test serum samples (50 μl) diluted 1 in 200 in blocking solution wereadded to test wells and incubated at 37° C. for 90 minutes.

“Test” plates were washed (3×) with saline/Tween 20.

Anti-rat conjugated HRP (RAM-HRP; 50 μl/well) diluted I in 1000 inblocking solution was added and the plates incubated under ambientconditions for 120 minutes.

Plates were washed (3×) with saline/Fween 20.

The reaction was initiated by addition of TMB (100 μl/well). Plates wereheld in a humid box for 60 minutes.

The reaction was terminated by addition of sulphuric acid (2.0 M; 50μl/well). Plates were held in a humid box for 60 minutes.

Absorbance readings were taken using a Multiscan plate reader usingFilter 1=4=450 nm and Filter 2=8=690 nm.

Results:

Observations

TABLE 4 x 10 17 24 01 08 15 17 Nov. Nov. Nov. Dec. Dec. Dec. Dec. Group1997 1997 1997 1997 1997 1997 1997 Mean Group A 0.097 0.094 0.121 0.1150.284 0.428 0.490 Mean Group B 0.095 0.131 0.518 0.612 0.681 0.677 0.622Stdevp Group 0.007 0.010 0.013 0.007 0.307 0.422 0.413 A Stdevp Group0.008 0.038 0.309 0.359 0.391 0.383 0.392 B t-test 0.65 0.02 0.00390.0025 0.028 0.21 0.5 significance values (p) The data set out above arerepresented graphically in FIG. 11 of the drawings.

Conclusions

These data show a marked delay in the seroconversion of rats in Group A,relative to those of Group B. When analysed using Student's t-Test, astatistically significant difference between the data for the two groupswas found at 7, 14, 21 and 28 days following administration of theformulations to the animals (ie, between Nov. 17, 1997 and Dec. 8,1997). Subsequent to Dec. 8, 1997, the immune response of the animals inthe respective groups showed no statistically significant difference,suggesting that seroconversion in Group A animals had simply beendelayed, and was reaching a similar response level to that observedearlier in Group B. The seroconversion of rats treated with thecontrolled release devices of the invention was therefore delayed—on astatistically significant basis—relative to the delay observed in theGroup B rats.

The claims defining the invention are as follows:
 1. A method ofproducing a delivery device for the controlled release of a bioactivesubstance, which comprises the steps of: (a) forming a generallyelongate structure by melt processing extrusion and extrusion whenheated of one or more pharmaceutically or veterinarily acceptablepolymeric materials so as to coat a core material containing thebioactive substance, the coating structure having an internal cavityextending at least substantially along its length, and wherein, theinternal cavity of the coating structure is capable of receiving thecore material; (b) inserting the core material into the internal cavityof the coating structure; (c) compressing the coating structure at afirst location along its length so as to generally form a seal at thatlocation; and (d) compressing the coating structure at a second locationalong its length.
 2. A method as claimed in claim 1, wherein the one ormore pharmaceutically or veterinarily acceptable polymeric materialscomprise materials which are degradable.
 3. A method as claimed in claim2, wherein the coating structure is formed as a single layer.
 4. Amethod as claimed in claim 2, wherein the coating structure comprisestwo or more layers.
 5. A method as claimed in claim 4, wherein the twoor more layers are layers of the same material.
 6. A method as claimedin claim 4, wherein the two or more layers are layers of two or moredifferent materials.
 7. A method as claimed in claim 1, wherein thepharmaceutically or veterinarily acceptable polymeric material ormaterials are selected from the group consisting of: (a) polylactideco-glycolide polymers; (b) polyesters; (c) polysaccharides; (d)polyamides; (e) poly (amino acids); (f) poly (ortho esters); (g)polyanhydrides; (h) polyphosphoesters; (i) polymers formed through thecombination of chemical bonds; and (j) combinations of two or more of(a) to (i).
 8. The method of claim 7, wherein the polymers of group (i)comprise: (a) pseudo-peptides; (b) poly (phosphoester-urethanes) (c)polydepsipeptides; and (d) combinations thereof.
 9. A method as claimedin claim 1, wherein the core material comprises: (a) a tablet; (b) agel; (c) a paste; (d) granules; (e) powder; (f) a fluid; or (g)combinations of two or more of (a) to (f).
 10. A method as claimed inclaim 1, wherein the core material comprises: (a) a bioactive substanceselected from the group consisting of: (1) natural, synthetic orrecombinant pharmacological agents; (2) food additives; (3) foodsupplements; (4) antigens; (5) antibodies; (6) cytokines; (7) growthpromotants; (8) hormones; (9) vaccines; (10) cancer cell inhibitoryagents; (11) immuno-suppressants; (12) immuno-stimulants; (13)antimicrobial agents; (14) anti-viral agents; (15) vitamins; (16)minerals; (17) organic nutrients; (18) inorganic nutrients; and (19)combinations of two or more of the aforegoing; and (b) optionally, oneor more pharmaceutically or veterinarily acceptable carriers and/orexcipients.
 11. A method as claimed in claim 1, wherein the step ofcompressing the coating structure at the first location along its lengthso as generally to form a seal at that location comprises the step ofbringing two or more mutually opposed surfaces of the coating structureinto contact with one another, so as generally to form a seal at thatlocation.
 12. A method as claimed in claim 11, wherein the step ofcompressing the coating structure at the first location along its lengthcomprises cutting the coating structure at that location.
 13. A methodas claimed in claim 1, wherein the step of compressing the coatingstructure at the second location along its length so as to generallyform a seal at that location comprises the step of bringing two or moremutually opposed surfaces of the coating structure into contact with oneanother, so as to form a seal at that location.
 14. A method as claimedin claim 1, wherein the step of compressing the coating structure at thesecond location along its length does not give rise to the formation ofa seal at that location.
 15. A method as claimed in claim 13, whereinthe step of compressing the coating structure at the second locationalong its length comprises cutting the coating structure at thatlocation.
 16. A method as claimed in claim 13, wherein a generallysealed container is formed.
 17. A method as claimed in claim 14, whereina container is formed which is generally sealed at one of two mutuallyopposed ends thereof, but not at the other end.
 18. A method as claimedin claim 1, wherein steps (a) to (d) are repeated along the length ofthe coating structure, so as to produce a plurality of coated structuresin the form of controlled release delivery devices for a bioactivesubstance.
 19. A method as claimed in claim 18, additionally comprisingautomating the repeated performance of the method along the length ofthe coating structure.
 20. The method as claimed in claim 1, wherein themethod comprises performing steps (a) to (d) sequentially.
 21. Themethod as claimed in claim 1, wherein the method comprises performingsteps (a) to (d) non-sequentially.
 22. The method as claimed in claim21, wherein said non-sequential steps are performed as follows: (i) step(a) is the first step; (ii) step (c) is the second step; (iii) step (b)is the third step; and (iv) step (d) is the fourth step.